1. Chitin and Chitosan.
Poly[β(1,4)-2-amino-2-deoxyglucopyranose] oligomers are known in the art as chitosan and its preparation and action as a nematicide has been abundantly discussed in specialized literature. It is also known to produce stimulatory action on chitinolytic microorganisms.
The term “oligomer” herein refers to a polymer with a low molecular weight; specifically, a chitosan polymer.
As it is well known, poly[beta(1,4)-2-amino-2-deoxyglucopyranose], hereinafter referred to as “Chitosan”, is produced by deacetylating the β(1,4)-2-acetamide-2-deoxy-D-glucose chitin, by hydrolysis thereof in an alkaline medium, usually NaOH or KOH, at high temperatures. This reaction and the resulting degree of deacetylation is sensitive to alkali concentration, particle size and solution density.
The term “deacetylation” as used herein refers to removal of one acetyl group (—CO—CH3) from every chitin ring.
The term “degree of deacetylation” or “deacetylation %” as used herein refers to the percent ratio between amino groups/amido groups in the chitosan polymer. Some colorimetric methods such as the one described by Roberts & Domsey use the following ratio % DA=(1−A1655/A3340×1/1.33)×1100, where A is the logarithmic ratio of absorbance to transmittance at a given wavelength.
Natural chitin can be found mainly in the shells of crustaceans and in the exoskeletons of insects, as well as in cell walls of many fungi such as ascomycota, zygomycota, basidiomycota and deuteromycota, and yeasts, and seaweeds such as diatoms. [Muzzarelli R. in “Chitin”. Pergamon Press. First Edition. 1974]. It is totally insoluble in water or in acid media. Additionally, native chitin is accompanied by inorganic salts, calcium carbonate being the most frequent among them, so it must be processed from its original matrix through successive deproteinization steps in an alkaline medium and demineralization in an acid medium.
Complete deacetylation of chitin results in a material that is totally soluble in an acid medium known as chitan. When deacetylation is incomplete, a mixture of chains is formed having different proportions of β(1,4)-2-acetamide-2-deoxy-D-glucose and β(1,4)-2-amino-2-deoxy-D-glucose units whose ratio depends on reaction conditions and therefore generate polymers with random molecular structures differing among them. [Lárez, Cristóbal. in “Algunos usos del Quitosano en sistemas acuosos”. Revista Iberoamericana de Polímeros, Vol 4(2), 2003]. Said differences include their length, the percentage of amino acetyl groups present and their positions along the chain. Chitin deacetylation procedures are known and available to those skilled in the art.
It is particularly preferred to use chitin deacetylase, the enzyme that catalyzes conversion of chitin into chitosan by deacetylating the residues of N-acetyl-D-glucosamide. This enzyme was first identified and partially purified from extracts of the Mucor ouxii fungus. Nevertheless, its use could result in reduced efficiency to deacetylate insoluble chitin substrates. This problem has been overcome by previously treating the crystalline substrates of chitin to ease access of acetamide groups to the enzyme.
Solubility in water of salt polyelectrolytes of chitosan in an acid reaction depends on the nature of anions involved, the degree of deacetylation, the molecular weight of the polymer and temperature. If deacetylation of the original chitin does not produce a ratio of at least 60% amino groups to amido groups in the polymer, it will continue to be insoluble in water, even if it is soluble in an acid to neutral medium due to protonization of the amino groups, given that they have a pKa value of about 6.5. In other words, chitosan is a bioadhesive and easily bonds with negatively charged surfaces, such as membranes. Chitosan improves transportation of polar molecules through epithelial surfaces. Nonetheless, some procedures are known in the art allowing production of water soluble chitosans, in spite of degrees of deacetylation with an amino/amido ratio below 60%. In fact, the maximum degree of deacetylation has been found to be 75-85%.
The yield of chitosan production is assessed by reaction with ninhydrin, determining the blue-violet staining of complexes formed between the amino groups of chitosan and the ninhydrin added to test tubes containing 1 ml of chitosan solution at different concentrations, and boiled for 10 min. Quantification is made by comparison with a calibration curve previously set to relevant ranges. This technique is described in Bohinnski, “Bioquímica”5th edition. Pearson, 1991; Van Hola, Mathew, “Bioquímica” 3rd ed, Pearson, 2004; and Feseden & Feseden, “Química Orgánica”, Interamericana, 2002; and is well known in the art.
Additionally, Chitosan has the property of biodegradability making it an especially preferred polymer material [Muzzarelli R. in “Chitin”. Pergamon Press. First Edition. 1974].
Chitosan oligomers offer many benefits over a large number of plant species, and there is abundant literature to support this fact. Usage of chitin and chitosan in agriculture is focused on improving the agronomical yields of several mechanisms. Seeds covered with chitosan solutions show improved sprouting and produce high yields when harvested. Horticultural products are frequently subject to mechanical damages, physiological alterations or attacks from pathogenic agents so using chitosan on these products results in a protective microbial activity and provides them with a cover generated by the filmogenic property of its solutions. These films are semipermeable to oxygen and carbon dioxide and also have proved to be antifungal, and therefore, they improve the quality and extend the useful life of treated fruits.
It has also been reported that an important benefit is the protection they provide on treated parts regarding attacks from bioantagonists such as bacteria, insects and nematodes.
Xiao Fei Liu et al in “Antibacterial action of chitosan and carboxymethylated chitosan” (Journal of Applied Polymer Science, vol. 79(7), 1324-1335, 2001) disclose the conclusions of applying chitosan with different molecular weights in order to assess the antibacterial action against E. coli in a lab culture containing meat extract, peptone and agar. The antibacterial action of chitosan is influenced by its molecular weight, the degree of deacetylation, its concentration in the solution and the pH of the medium. In particular, the test shows that water soluble chitosan produces a good antibacterial action against E. coli which increases with a molecular weight between 5000 and 9.16×104 Da, and decreases when molecular weight of the polymer increases to between 9.16×104 and 1.08×106 Da. Besides, the antibacterial activity of oligomers (polymers with a low molecular weight) has been proved to be caused mainly by inhibiting DNA transcription.
El Ghaouth, Ahmed et al, of Laval University, Quebec, Canada have published several scientific works showing the antifungal activity of chitosan. In “Efecto de la Aplicación de Quitosano para controlar Botrytis en Pimentón” (Physiol. and Molec. Plant Pathology, vol. 44, 417-432, 1994) they communicate the results of treating cuts in the peduncles of peppers with chitosan solution that were later infected with spores of Botrytis cinerea. It could be found that fruits treated with chitosan did not exhibit a visible disease until 7 days after inoculation, instead, in control fruits the disease was visible within 24 hours. A further review made 14 days later showed that all control fruits were infected, whereas only 25% of fruits treated with chitosan were infected.
In “Efecto de la Aplicación de Quitosano in-vitro a hongos patógenos” (Phytology Department, Laval University, Quebec, Canada) the antifungal effect of chitosan was researched through the growth (in-vitro) of common pathogenic fungi in postharvest strawberries. Research showed that chitosan substantially reduces the radial growth of Botrytis cirenea and Rhizopus stonolifer to great effect under high concentrations. These authors also confirmed the significance of the amount of positively charged groups (−NH3+) along the polymer chain.
In “Efecto de la Aplicación de Quitosano para Controlar Pythium e inducir reacciones de defensa en Pepinos” (The American Phytopathological Society, Vol. 84, 313-320, 1994) the authors grew a cucumber plant contaminated with Pythium aphanidermatum in a solution with nutrients to which chitosan was added. As a result, root putrefaction could be controlled and a number of defensive responses were activated, including induction of structural barriers in root tissues and stimulation of antifungal enzymes in both roots and leaves, without causing any residual toxic effects in the plants.
Benhamou, Nicole and Theriault, George in “Efecto de la Aplicación de Quitosano para el control de Fusarium en Tomate” (Physiol. and Molec. Plant Pathology, 33-52, 1992) disclosed the results of applying chitosan to tomato plants before inoculation with the root pathogen Fosarium oxysporum f. sp. Radicis-lycopersici. Research showed that chitosan effectively protected the plant against putrefaction of the neck and root. It also showed that it is not only useful in stimulating the general defense system of plants but also in reducing the effect of prevalent pathogens in the soil such as Fusarium. Protection against fungal attack on the roots could be observed for more than 6 days.
Concerning the present invention, the nematicide activity property of these polymers is preferred. It has been shown that said nematicide activity is effective in species such as vegetables, asparagus, pulses, cereals, oilseeds, beetroot, cotton, tobacco, fruit plants, ornamental plants, woods, tomatoes and peppers. The mechanism of protection with nematicide activity is attained by inducting a physical barrier in seeds as well as in plant roots, even though there also exist other mechanisms such as activating resistance genes, activating proteins connected with resistance responses, or activating chitinase or accumulation of physaline, an antibiotic and antifungal, or a combination thereof.
Carrera, L. et al in “Efecto Nematicida de Enmiendas de Quitina y Quitosano sobre el Nematodo Nodulador” (Polymer Lab, National University, Costa Rica) discloses performance of replicated tests (×6) in soils affected by nematodes of the Meloedogyne sp. type in experimental tomato crops (Lycopersicon esculentum MILL, Hayslip variety) in pots, with three weeks of vegetative growth, applying doses of 0, 0.2, 0.4, 0.8, 1.0 w/w of chitin and chitosan. Some significant parameters of nematicide activity were assessed, such as plant height, fresh weight of roots, number of galls in roots, gall index, number of nematode infective stages (J2) in 100 g of soil, number of nematode eggs in 25 g of root, and pH. In the conclusions they reported that the number of infective stages of Meloedogyne sp. and the number of eggs by root weight as related to increasing doses of chitin were highly significant. 0.8% and 1% doses of chitin were very effective and comparable to conventional chemical treatment, whereas chitosan in a dose of 0.8% showed a clear tendency to control the microorganism.
Particularly relevant and setting an important precedent was the experimental assay developed in order to comply with the requirements for registration of the BIOROOT (NEMATICID)® product, owned by the inventors of the present invention, before the Ministry of Cattle, Agriculture and Fisheries of Uruguay. In said experimental work, they performed a “comparative research of poly[beta(1,4)-2-amino-2-deoxyglucopyranose] as a stimulant for root growth and as a protective agent from nematode colony growth” (Lopretti, Mary et al, 2006) in greenhouse tomato crops (Lycopersicom esculentum) of the hybrid NIXE variety and hybrid pepper (Capsicum anum), autumn-winter cycle, and assessing the results using statistical techniques known to those skilled in the art. A 2% chitosan solution produced from chitin was used, in order to verify the root growth action together with the protective and nematostatic action. This work concluded that a stimulating effect of root growth showed in plants treated with chitosan, wherein an increase in root width and mostly in root length could be confirmed. This increase allows for improved anchoring, improved exploration ability and improved water and nutrient absorption, which is made apparent by larger leaf areas, with larger fruits than control plants. Furthermore, it was concluded that using these natural polymers, both chitin and chitosan, formation of chitinolytic agents is enhanced.
In brief, and taking into account the scope of the present invention, usage of chitosan, a natural, biodegradable and non-toxic polymer of a cationic type, protects treated parts of soils, plants and parts of plants, such as roots, stems, leaves, seeds, flowers and fruits, more preferably roots, from the attack of bioantagonists such as bacteria, fungi, insects and nematodes. This provides the plant with greater tolerance to neck and root health problems increasing the vitality of plant cells, accelerating degradation of cell walls in fungi and organisms containing chitin in their structure, delivering additional chitosan in the process. In addition to this, it stimulates plant cells to produce biochemical compounds that strengthen cell walls, thus significantly improving resistance to stress situations, drought, excessive dampness, transplants and frost. An increase in root mass is triggered, which translates into an increase in the speed of growth and greater plant vigor.
2. Lignin
Lignin is an aromatic, non carbohydrate complex, of which many structural polymers exist. It is a macromolecule, with a high molecular weight resulting from the union of phenylpropyl alcohols: cumaryl, coniferyl and sinapyl whose structures are as follows:

Randomized coupling of these molecules produces a three-dimensional, polymeric and amorphous structure, depending on the source of raw material for lignin production. This is why it is not possible to describe a definite structure, although there is a plurality of models providing approximate representations of said structure, one of which is illustrated below by way of example:

However, for the purposes of the present invention, this fact is not relevant. They are insoluble in acids and soluble in strong alkalis, such as NaOH.
Lignin is the main constituent of secondary wall cells in plant fibers, providing rigidity for structural support, and impermeability for water transport. After cellulose, this polymer is the most abundant organic compound in the biosphere. In different species of trees, lignin contents vary between 15 and 36% of dry wood material.
Processes to produce modified lignin phenols have been described and are known in the art.
Particularly, patent application UY26.985 (Uruguay) filed on Oct. 25, 2001, by the same inventor of the present invention, and incorporated by reference herein in its entirety, discloses a method to produce it using biotechnological means based on the fact that some microorganisms, such as fungi, bacteria, etc. are able to modify lignin. For the scope of the present invention, it is preferred that such microorganisms be fungi, including the following genera: Coriolus spp, Phanerochaetes spp, or Gloeophylum spp, in whose enzymatic extracts the following enzymes can be found: Lig-peroxidase, Mn-peroxidase, Poliblue-oxidase, demethylases, and oxygenases, among others. Particularly preferred are so-called brown rot fungi, such as Gloeophyllum trabeum, and so-called white rot fungi, such as Phanerochaete chrysosporium. Both types of fungi demethoxylate lignin producing hidroxyl groups in phenolic or non-phenolic structures, and produce oxidation of lateral chains and cleavages in aromatic rings. Brown rot fungi produce their main effect by demethoxylating aryl methoxyls, thus increasing the number of phenols. White rot fungi are characterized by depolymerizing lignin with a Lig-peroxidase or Mn-peroxidase, and they are the only known organisms able to degrade it completely into carbon dioxide and water.
In the present invention, it is preferred that this component of the biocidal composition be a mixture of oxidized natural phenols with a low molecular weight.
The production process described in UY26.985 includes the following steps: characterizing lignin, preparing the enzymatic biological agent by culturing fungi or obtaining enzymatic extracts from these fungi; depolymerizing lignin in a biological fermenter under controlled conditions; separating the phases, where the solid phase is obtained by precipitating lignins with a high molecular weight; filtering; extracting soluble modified phenols with centrifugation; purifying modified phenols with precipitation; characterizing and controlling the quality of the final product of the mixture of modified phenols whose molecular weight is less than 800; and controlling bactericide and fungicide activity.
In this method, the phenol hydroxide contents were analyzed according to the method described by Goldschmid, well known in the art, where a reference calibration curve of lignin is used vs. absorbance at 280 nm on samples obtained 0 hours after enzymatic fermentation and up to 192 hours after it began. Biomass changes during fermentation were analyzed by determining the weight/volume ratio of fermentation media 0 hours and 144 hours after fermentation began.
The term “natural phenols” as used herein refers to phenols from a plant source obtained with the strategy of enzymatic biotechnological production disclosed in UY26.985, where demethoxylation of phenols occurred exclusively by the oxidizing action of the enzymes of fungi and bacteria. The term “natural phenols” is used as opposed to other phenols that are produced by organic synthesis in labs or derived from oil.
For the purposes of the present invention, it is a relevant fact that phenols used in the biocidal composition of the invention that will be described below should be natural using the enzymatic processes of fungi and bacteria which can be found in the vicinity of the plant to be protected and in surrounding soil.
The term “modified phenols” as used herein refers to phenols oxidized by the above mentioned enzymatic processes.
The term “demethoxylation” as used herein refers to removal of one or two methoxide groups (—O—CH3) linked to the phenol ring, substituting it for a —H.
Under the conditions described in the paragraphs above, a final product is obtained, which is biocidal, enhancing plant resistance to phytopathogenic agents and which is based on modified natural phenols from plant sources that provide protective activity against said phytopathogenic agents, and which are particularly and preferably interesting for the composition of the present invention.
3. State of the Art
In concordance with the description under “Background of the Invention”, the state of the art shows that chitosan, with its disclosed properties and marketed products containing it, provides an excellent ability to control bacteria, insects and nematodes, especially nematodes, by increasing chitinolytic flora, but concomitantly it shows an inefficient fungicide activity.
On the other hand, Lignin shows an efficient fungicide action against phytopathogens such as Botritis, Esclerotinia, etc. and facilitates transformation of organic materials in soils, but has no fungicide action on fungi affecting lignin and has a very inefficient bactericide activity.
No composition for agricultural use has been disclosed meeting the requirements to obtain in a single composition the synergy of a disinfecting action against soil fungi and bacteria together with bacteriostatic and fungistatic activity and bactericide, fungicide, microbicide and nematicide activity, all of them provided in agronomically efficient doses.